Summary The objective of the proposed work is to identify mechanisms that allow vascular endothelial growth factor A, VEGF, to regulate intracellular processes without being secreted (intracrine signaling), in addition to controlling cell and tissue functions by receptor-mediated pathways (paracrine signaling) as a secreted protein. In previous studies, the investigators developed evidence in support of the conclusion that VEGF controls development, postnatal growth and homeostasis of bone by both signaling pathways. Furthermore, experiments with cells in culture and studies of mice with VEGF-related genetic modifications generated novel insights into structural and functional differences between secreted and intracellular forms of VEGF in bone- forming cells. The planned experiments are designed to increase understanding of how intracellular VEGF functions are regulated in skeletal tissues. Furthermore, intracellular processes, controlling the differentiation of skeletal progenitor cells into either bone-forming osteoblasts or bone marrow adipocytes, will be examined in detail. A better understanding of these processes is likely to lead to additional research of substantial clinical value. Anti-VEGF drugs, now in clinical use for treatment of age-dependent eye disease and cancer, are targeting paracrine VEGF signaling and are therefore unable to affect processes inside cells that produce the growth factor. In addition to its role in differentiation of bone-forming cells, intracellular VEGF has been found to have important functions as a survival factor for cancer cells, an agonist in cardiovascular homeostasis, and stimulator of hematopoietic stem cell differentiation. Thus, by elucidating intracellular VEGF-dependent mechanisms, the data generated by the proposed studies will likely provide a basis for novel therapeutic strategies aimed at modulating such functions.